Introduction

Heart failure (HF) is a primary cause of morbidity and mortality worldwide and characterized by left ventricular (LV) hypertrophy, cardiac fibrosis, and inflammation that results in systolic and diastolic dysfunction.1,2 The accumulation of collagen and other extracellular matrix proteins in the cardiac interstitium can also disrupt the cardiac cycle, causing profound functional impairment.3 Mechanical cardiac overload is accompanied by various physiological changes, including myocyte apoptosis, inflammatory and fibrotic responses, hypertrophy, and angiogenesis, among others.4 Of these, accumulating evidence suggests that proinflammatory cell infiltration in the myocardium plays a pivotal role in the initiation and development of cardiac remodeling and dysfunction.5,6

Platelets were originally thought to serve a vital role in hemostasis and thrombosis; however, recent studies demonstrate their function in the inflammatory and immune responses via proinflammatory cytokine release and interactions with endothelial cells, leukocytes, and smooth muscle cells.7,8 Platelets normally circulate in a quiescent state and are sensitive to circulation changes resulting from endothelial cell damage, sheer stress conditions, and hemodynamic abnormality.9,10 On activation, these cells change their morphology and release cytokines, chemokines, and growth factors stored within granule compartments to regulate pathological cardiac remodeling11,12 and upregulate specific adhesion molecules necessary to interact with other immune cells, such as P-selectin/PSGL-1 (P-selectin glycoprotein ligand 1).8,13,14 Similarly, activated platelets also bind to endothelial cells to induce their expression of various adhesion molecules which subsequently promote leukocyte recruitment.15

Materials and Methods

Ethics Statement

All procedures were approved by the Experimental Animal Research Committee of Tongji Medical College, Huazhong University of Science and Technology, and performed according to the recommendations of the Guide for the Care and Use of Laboratory Animals (National Institutes of Health [NIH] Publication No. 85-23, revised 1996).

Animal Models

P2y12−/− mice (C57BL/6) were provided kindly by Dr Jun L. Liu23 and housed in the specific pathogen-free animal center of Tongji Hospital. Transverse aortic constriction (TAC) or sham surgeries were performed in 8- to 12-week-old male P2y12 wild-type (WT) and knockout (KO) mice by ligating the aorta with a 27-gauge needle between the first and second branch of the aortic arch as described previously.24 The pressure gradient over the aortic ligature was determined using color Doppler, and only mice with a pressure gradient ranging from 50 to 70 mm Hg were used in further experiments.

For bone marrow (BM) transplantation, BM cells were isolated from WT or P2y12 KO mice as previously described.25 Subsequently, 8- to 12-week-old WT recipient mice, pretreated with polymyxin and neomycin for 14 days, were lethally irradiated with 8.5 Gy, and then injected with 5×106 BM cells within 4 hours. Recipient mice were allowed to recover for 4 weeks before subjected to TAC or sham surgery.

Platelet Reconstitution

Platelets (5×108) isolated from WT mice were injected into the tail vein of KO BM recipients the day before TAC surgery. Injections were repeated every 5 days for 4 weeks as previously reported.26

Statistical Analysis

All data represent the mean±SEM from at least 3 independent experiments. Comparisons between 2 groups were performed with 2-tailed Student t testing for parametric data. For comparisons of >2 groups, a 1-way ANOVA, followed by post hoc testing using Bonferroni correction, was used to determine differences between the experimental conditions. P<0.05 was considered statistically significant.

Results

To examine systemic P2y12 receptor expression, tissues from 9 different organs were collected from WT C57BL/6 mice and assessed by Western blotting. Results showed that the P2y12 receptor was highly expressed in brain and lung, but rarely in kidney, aorta, heart, liver, and muscle (Figure S1A in the online-only Data Supplement). Subsequent immunofluorescence of cardiac tissue revealed that the P2y12 receptor was mostly expressed on vascular smooth muscle cells (Figure S1B), but was unaffected by changes in blood pressure (data not shown). In addition, P2y12 analysis by flow cytometry demonstrated high levels of P2y12 staining on platelets, but negligible expression in other leukocyte subsets (Figure S1C).

Pressure Overload Elicits Platelet Activation and Deposition in Heart

To investigate the effect of pressure overload on platelet physiology, WT mice were subjected to TAC or sham surgery, and then platelet activation was monitored by flow cytometry. Notably, a marked increase in platelet activation was observed in WT mice 3 days after TAC (31.61±6.07%), which gradually declined at 1 week (27.89±3.78%) and 4 weeks (20.96±3.24%) post-surgery (Figure 1A); however, no significant changes were observed in P2y12 KO mice.

A previous study reported that platelet accumulation increases in infarcted hearts.21 As expected, CD41+ cell proportion displayed a significant increase in WT hearts compared with KO counterparts by immunostaining, peaking at 1 week after TAC operation (Figure 1B). This was confirmed by Western blot (Figure 1C). Moreover, the expression of P2y12 mRNA in heart was significantly increased at 4 weeks post-TAC operation compared with sham group (Figure S1F). These data suggest that P2y12-mediated platelet activation may play an important role in pressure overload–induced myocardial remodeling.

Hemodynamic and echocardiographic data are shown in Figure 2F and Table S1. Although there were no significant differences in systolic and diastolic function (dP/dtmax and dP/dtmix), LV fractional shortening and ejection fraction, the increases of left ventricular internal diameter and left ventricular posterior wall thickness in WT mice were reversed in P2y12 KO at 1-week post-TAC. Four weeks after operation, both dP/dtmax and dP/dtmix were significantly improved in P2y12 KO mice compared with WT TAC counterparts (Figure 2F). Moreover, the increase of LV diameter and wall thickness and the decrease of LV fractional shortening and ejection fraction in WT TAC mice were restored in P2y12 KO TAC mice (Figure 2F). Collectively, these data indicated that systemic P2y12 deficiency attenuates pressure overload–induced myocardial remodeling and cardiac dysfunction.

Because P2y12 is highly expressed in platelets, but rarely in other BM-derived cells,31 we designed a reconstitution experiment to investigate the role of activated platelets in cardiac hypertrophy. Notably, administration of WT platelets to P2y12 KO chimeras rescued the defects in platelet activation and aggregate formation (Figure 5B through 5E; Figure S5A through S5D). These mice also exhibited a similar clinical presentation that observed with WT TAC mice (Figure 4A through 4E; Table S2). Moreover, immunohistochemical analysis of the hearts indicated that neutrophil and macrophage accumulation was significantly increased by WT platelet reconstitution compared with P2y12 KO chimeric mice, but no statistical difference was found in the number of CD3+ lymphocytes (Figure 5A).

Despite corresponding changes in platelet–leukocyte aggregation, it was still obscure that whether the change is the cause of the effects or just an accompanying phenomenon. As previously described, binding of P-selectin on the activated platelet membrane to leukocyte PSGL-1 is a dominant molecular event, although other adhesive mechanisms exist.32,33 Thus, we used P-selectin neutralizing antibody to inhibit the interaction between platelets and leukocytes. As expected, P-selectin blockage resulted in a significant reduction in aggregate formation when compared with IgG controls after surgery (Figure 6E), and decreased hypertrophy and fibrosis (Figure 6A through 6C), inflammatory cell infiltration (Figure 6D; Figure S6A), and a partial increase in systolic and diastolic function (Table S3).

Although our research suggests that activated platelets promote myocardial remodeling by triggering the inflammatory cascade, whether there exists a direct promoting effect on cardiomyocyte hypertrophy or fibroblast activity remains unclear. As such, we used ADP to stimulate platelet α-granule release, whereas anti-NSF (N-ethlymalimide-sensitive factor) was used to block exocytosis. As shown in Figure S6C, the concentration of PF4 (platelet factor 4)—a marker of α-granule release—was significantly increased in the supernatant of ADP-stimulated WT platelets when compared with that from unstimulated controls and was mitigated by P2y12 KO or anti-NSF treatment. As shown in Figure S7A and S7B, activated WT platelets caused 1.7-fold change on myocyte size when compared with unstimulated controls, although P2y12 KO platelet or fixed platelet caused limited change. Similar results were observed with anti-NSF antibody. Moreover, expression of the fibrotic markers α-SMA (α-smooth muscle actin) and collagen I on NIH/3T3 fibroblasts showed an identical trend (Figure S7C through S7E). Therefore, we concluded that P2y12-mediated platelet α-granules release modulates cardiac hypertrophy and fibrosis.

Discussion

Previous studies show that TAC is a critical regulator of myocardial function, gene expression, and physiological changes, including increased fibrosis, inflammation, and cardiomyocyte apoptosis.2 Various anti-inflammatory strategies have been proposed to treat TAC-induced myocardial remodeling and cardiac dysfunction. Interestingly, many findings in our study highlight an essential role of P2y12-mediated platelets activation in pressure overload–induced cardiac remodeling, inflammation and dysfunction.

Increasing evidence suggests that patients with hypertensive hypertrophy show abnormal platelet activation. Liu et al21 and Gurbel et al34 both reported that myocardial infarction induced platelet activation and deposition, which was responsible for local and systemic inflammation, leading to cardiac remodeling. Moreover, platelet activation and deposition were also observed in angiotensin-II infusion-induced inflammation and fibrosis.35 However, whether the beneficial effects of antiplatelet strategy could be confirmed in pressure overload–induced myocardial remodeling remained unknown. Interestingly, we observed a significant increase in CD62P+ platelets and platelet deposition in TAC mice, but not in P2y12 KO or P2y12 KO chimera counterparts, suggesting that P2y12-mediated platelet activation contributed to pressure overload–induced myocardial remodeling.

Activated platelets are known to release various proinflammatory mediators stored within their granule compartment.36 Besides, activated platelets trigger secretion of more proinflammation mediators from endothelial cells, leukocytes, or smooth muscle cells after an intimate contact.14,32,37 Previous reports revealed that treatment with the specific P2y12 antagonist clopidogrel blocked increases in serum CD40 ligand, C-reactive protein, and P-selectin.38 Moreover, long-term clopidogrel therapy was proven to significantly diminish plasma concentrations of various inflammatory cytokines in subjects who underwent percutaneous coronary intervention, compared with clopidogrel-naive patients.22 We also observed elevated levels of mRNA in myocardium and serum protein expression of various proinflammatory mediators in WT TAC mice, which was inhibited in P2y12 KO TAC mice. Collectively, these data indicated that systemic P2y12 KO produced stronger anti-inflammation effect than clopidogrel treatment because clopidogrel was rapidly metabolized in vivo. More interestingly, our studies confirmed that P2y12-mediated platelet exocytosis directly affects cardiomyocyte hypertrophy and fibroblasts activity.

Platelet activation also stimulates surface expression of P-selectin (CD62P), a dominant molecule that mediates the interaction between leukocytes and platelets.33 Elevated levels of platelet–leukocyte complexes were detected in patients with unstable angina, acute myocardial infarction, those undergoing percutaneous coronary intervention, and in patients with peripheral arterial disease.8,13,14,19,39 Sreeramkumar et al32 suggested that neutrophils extended a PSGL-1–bearing domain into the vessel lumen to scan for activated platelets in the bloodstream through P-selectin binding, which provided a rapid and an efficient regulatory mechanism for the adhesion and rolling of neutrophils on the inflamed vasculature and migration to the lesion sites. Moreover, activated platelets also were reported to preferentially conjugate with proinflammatory CD16high monocytes,13 thereby accelerating proinflammatory mediators secretion and adhesion to vascular endothelial cells.40 In our research, we found that systemic or platelet-specific P2y12 deletion reduced TAC-induced MPO+ neutrophils and CD68+ macrophages infiltration possibly by decreasing platelet–neutrophil and platelet–monocyte aggregate in a P-selectin/PSGL-1–dependent manner. Consistently, platelet depletion and P-selectin blocking experiments further confirmed those conclusions. However, earlier studies had showed that P2y12 was detected in rat alveolar macrophages41 and contributed to macrophages navigating in a gradient of C5a by promoting lamellipodia formation.42 These studies indicate that the P2y12 receptor possibly had a direct effect on macrophage migration.

Healthy vascular endothelium is a barrier that prevents circulating inflammatory cell escaping from vessel. However, patients with progressive or acute HF always presented endothelial dysfunction.43 Previous studies revealed that clopidogrel and other antiplatelet treatments were thought to improve endothelial function.44,45 Our research found that P2y12 deficiency restrained the increase of ICAM-1, VCAM-1, P-selectin, E-selectin, and MCP-1 induced by TAC in proximal aorta. Similarly, in vitro platelets activated by the P2y12-specific agonist (2-Mes-ADP) stimulated endothelial cells to upregulate all those molecules. Our results indicated that the importance of vascular inflammation in pathological myocardial hypertrophy raised the intriguing possibility that antiplatelet agents imparted inhibitory effects on the platelet-driven inflammatory cascade and inflammation-related myocardial remodeling.

Perspectives

Congestive HF is accompanied with increased leukocyte accumulation and inflammatory cytokine expression in hearts, which exacerbate the development and progression of congestive HF. Activated platelets initiate and accelerate leukocyte activation and migration via immediate interaction. In the article, we demonstrate that P2y12 deficiency or platelets depletion attenuates pressure overload–induced cardiac inflammation and remodeling. In addition, the results from P-selectin blockage imply that platelet–leukocyte interaction contributes to pressure overload–induced inflammatory cell infiltration in hearts and the resulting remodeling. Our results suggest that strategies targeted to prevent platelet activation or platelet–leukocyte interaction may have potential use in treatment or control of congestive HF.

Acknowledgments

We thank Dr Junling Liu for providing the P2y12−/− mice and Drs Xingxu Wang and Yunzeng Zou for their help with animal echocardiography.

Sources of Funding

This work was supported by Projects from National Nature Science Foundation of China (No. 91439203 and 81630010), the National Basic Research Program of China (No. 2012CB518004), and the Fundamental Research Funds for the Central Universities (No. 2015ZDTD044).

. The role of the CXC chemokines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer.Cytokine Growth Factor Rev. 2011;22:1–18. doi: 10.1016/j.cytogfr.2010.10.011.

. Effects of reteplase and alteplase on platelet aggregation and major receptor expression during the first 24 hours of acute myocardial infarction treatment. GUSTO-III Investigators. Global Use of Strategies to Open Occluded Coronary Arteries.J Am Coll Cardiol. 1998;31:1466–1473.

. Further peripheral vascular dysfunction in heart failure patients with a continuous-flow left ventricular assist device: the role of pulsatility.JACC Heart Fail. 2015;3:703–711. doi: 10.1016/j.jchf.2015.04.012.

Novelty and Significance

What Is New?

This is the first study to demonstrate that P2y12-mediated platelets activation contributes to cardiac remodeling by triggering a series of inflammatory changes and interacting with leukocytes and endotheliocytes in congestive heart failure.

Summary

P2y12-mediated platelet-derived inflammation cascade, including platelet–leukocyte complexes and platelet–endothelial interaction, granule release, and may be the underlying mechanism of the observed beneficial effect.